Fluorocarbon-Linked Peptide Formulation

ABSTRACT

The invention provides an aqueous acidic formulation suitable for use as in the preparation of a pharmaceutically acceptable fluorocarbon-linked peptide formulation, which aqueous formulation comprises a first fluorocarbon-linked peptide, wherein: the peptide linked to the fluorocarbon is at least 20 amino acid residues long, comprises at least 50% hydrophobic amino acid residues and has an isoelectric point greater than or equal to 7; and the fluorocarbon-linked peptide is present in micelles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/977,265, filed Jun. 28, 2013, which is a U.S. national phaseapplication under 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/GB2011/001781, filed Dec. 30, 2011, which claims priority to andthe benefit of United Kingdom Patent Application No. 1022147.1, filedDec. 31, 2010, the contents of each of which is incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The invention relates to pharmaceutically acceptable formulationscomprising fluorocarbon-linked peptides, formulations useful in thepreparation of such pharmaceutically acceptable formulations, a methodof preparing such formulations and the use of such formulations asvaccines and immunotherapeutics.

BACKGROUND TO THE INVENTION

Synthetic peptide antigens are of interest for use in vaccines toprevent infectious diseases (such as viral, bacterial, parasitic andfungal infections). Synthetic peptide antigens are also of interest inthe field of immunotherapeutics, including the treatment of infection,the stimulation of immunity to cancer cells, the down-regulation ofpolypeptide hormones and the control of inappropriate immune responsessuch as anaphylaxis and allergy.

One difficulty in the practical use of peptide-based vaccines andimmunotherapies is ensuring the induction of an immune response byefficient delivery of the peptide antigens to an antigen presentingcell. Without such targeting, unfeasible amounts of the peptide may berequired, which would not only be uneconomical to manufacture but couldalso lead to toxicity issues.

Enhancement of peptide delivery may be achieved through specialiseddelivery vehicles, such as particulate-based structures enablingsustained release. In addition, peptide derivatives or modified peptidescomprising the peptide of interest covalently linked to adelivery-enhancing agent have been developed to improve thebio-availability and presentation of the peptide to specific targetcells and receptors.

One particular class of peptides modified to improve delivery to antigenpresenting cells are constructed through the covalent attachment of afluorocarbon chain to either the peptide N- or C-terminus, or at anyposition in between, to create a fluorocarbon-linked peptide (FCP).Examples of fluorocarbon-linked peptides are given in WO2005/099752 andWO2009/027688 and the advantages afforded by the fluorocarbon attachmentin the enhancement of immune responses to the peptide are providedtherein.

It will be understood by vaccine designers that more than one peptidemay be required to provide a broader prophylactic or immunotherapeuticeffect. Such multi-component products are desirable since they arelikely to be more effective at eliciting appropriate immune responses.In order to manufacture a pharmaceutical product of this nature, thefluorocarbon-linked peptides must be synthesised, purified, blendedtogether at appropriate ratios, rendered sterile and presented in ahomogenous format suitable for administration.

SUMMARY OF THE INVENTION

The present inventors have found that fluorocarbon-linked peptides areoften poorly soluble in aqueous media, such as water or phosphatebuffered saline, even when the unlinked peptides are soluble in aqueousmedia. They have further found that the length and hydrophobicity of thepeptide component of the fluorocarbon-linked peptide affects thesolubility of the fluorocarbon-linked peptide. In particular,fluorocarbon vectors linked to longer, more hydrophobic peptides thatdisplay better immunogenetic properties have been found to beparticularly insoluble.

Fluorocarbon-linked peptides are amphiphilic and characteristically formmultimolecular micellar-type structures in both polar (protic andaprotic) and non-polar solvents. Such structures are not typicallyformed by native unlinked peptides. However, the inventors have foundthat many fluorocarbon-linked peptides, especially those with the bestimmunogenic properties, have a tendency to form large visible aggregatesin aqueous media and other solvents. The formation of such aggregates isunacceptable in a pharmaceutical manufacturing process, which requiresthe production of a homogeneous, characterisable formulation.

Having identified this problem, the present inventors have addressed itand devised a method for preparing fluorocarbon-linked peptideformulations in which supra-molecular structures that support thesolubility of the fluorocarbon-linked peptides are maintained. Theformulation process developed by the inventors makes it possible tomanufacture a stable product comprising the immunogenicfluorocarbon-linked peptides that they have found to be problematic toformulate, which product is easy to reconstitute with an aqueous mediumto obtain a pharmaceutically acceptable solution.

In particular, the inventors have found that using an acidic solutionpromotes micelle formation and avoids the formation of insolubleaggregates. The solubilised fluorocarbon-linked peptides can besterilised by filtration without loss of the fluorocarbon-linkedpeptides from the solution. After freeze drying, typically in thepresence of a cryoprotectant, the fluorocarbon-linked peptides can bestored in a stable form and dissolved in an aqueous medium to obtain apharmaceutically acceptable solution for administration.

The inventors have found that acetic acid is a particularly appropriatesolvent for a wide range of fluorocarbon-linked peptides, despite thehigh degree of variability in charge and hydrophobicity of the differentpeptides. Acetic acid is therefore also particularly suitable forsolubilising a mixture of fluorocarbon-linked peptides.

Accordingly, the invention provides an aqueous acidic formulationsuitable for use as in the preparation of a pharmaceutically acceptablefluorocarbon-linked peptide formulation, which formulation comprises afirst fluorocarbon-linked peptide, wherein:

-   -   (i) the peptide linked to the fluorocarbon is at least 20 amino        acid residues long, comprises at least 50% hydrophobic amino        acid residues and has an isoelectric point greater than or equal        to 7; and    -   (ii) the fluorocarbon-linked peptide is present in micelles with        a diameter of less than 0.22 μm.

Preferably, the formulation comprises acetic acid. The aqueousformulation may have, for example, a pH of 5 or less.

The formulation may comprise one or more further fluorocarbon-linkedpeptide present in micelles with a diameter of less than 0.22 μm.Preferably at least 80% of the fluorocarbon-linked peptide micellespresent in the formulation have a diameter of less than 100 nm.

In one embodiment, the formulation according to the invention does notcomprise a fluorocarbon-linked peptide in which the peptide linked tothe fluorocarbon: (i) has an isoelectric point of less than 7; (ii) doesnot comprise a positively charged amino acid in the last 15 contiguousamino acids distal to the fluorocarbon; and/or (iii) comprises acontiguous sequence of 20 amino acid residues comprising more than 80%hydrophobic amino acid residues.

The peptides linked to the fluorocarbons are typically immunogenicpeptides derived from a pathogen, autologous protein or tumor cell. Theformulation according to the invention may further comprise apharmaceutically acceptable carrier or diluent and/or an adjuvant.

The invention also provides:

A method for obtaining a pharmaceutically acceptable fluorocarbon-linkedpeptide formulation, said method comprising:

(i) solubilising a fluorocarbon-linked peptide in acetic acid;

(ii) filter-sterilising the solubilised fluorocarbon-linked peptide; and

(iii) drying the filter-sterilised fluorocarbon-linked peptide.

a fluorocarbon-linked peptide formulation obtainable by a methodaccording to the invention;

a pharmaceutically acceptable formulation comprising sixfluorocarbon-linked peptides, wherein the peptides linked to thefluorocarbons comprise the sequences set out in SEQ ID NOs: 1 to 6 andwherein the formulation comprises no other fluorocarbon-linked peptides;

a pharmaceutically acceptable formulation according to the invention usein a method of treatment of the human or animal body by therapy;

a pharmaceutically acceptable formulation according to the invention foruse in a method of treating or preventing a pathogenic infection, anautoimmune disease or cancer;

use of a pharmaceutically acceptable formulation according the inventionin the manufacture of a medicament for treating or preventing apathogenic infection, an autoimmune disease or cancer; and

a method of treating or preventing a pathogenic infection, an autoimmunedisease or cancer, said method comprising administering to an individualin need thereof an effective amount of a pharmaceutically acceptableformulation according to the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an example of a typical fluorocarbon-linked peptidemanufacturing process flow.

FIG. 2 shows an alternative fluorocarbon-linked peptide manufacturingprocess flow.

FIG. 3 is a table showing the results of a visual examination ofindividual FCPs solubilized in various solvents. Following vortexingeach solution was visually examined for clarity, with a score of “+++”assigned for a clear solution through to a score of “−” for a highlycloudy solution. The degree of foam formation and the presence ofparticulates were also recorded, with “+++” indicating high levels and“−” indicating absence of each. “*” indicates that the solution becameviscous.

FIG. 4 is a table showing the results of a visual examination ofindividual FCPs solubilized in various solvents after dilution with amannitol solution. Each solution was visually examined for clarity, witha score of “+++” assigned for a clear solution through to a score of “−”for a highly cloudy solution. The degree of foam formation and thepresence of particulates were also recorded with “+++” indicating highlevels and “−” indicating absence of each.

FIG. 5 shows the particle size of fluorocarbon-linked peptides insolution post-blending and dilution assessed by Dynamic Light Scattering(DLS).

FIG. 6 shows the size and shape of fluorocarbon-linked peptide particlesin solution post-blending and dilution assessed by transmission electronmicroscopy (TEM).

FIGS. 7A and 7B show the size distributions by volume offluorocarbon-linked peptide particles before (FIG. 7A) and after (FIG.7B) sterilizing grade filtration assessed by DLS.

FIGS. 8A and 8B show the size distributions by volume offluorocarbon-linked peptide particles reconstituted in Tris 10 mM pH7.85 (FIG. 8A) and water (FIG. 8B) assessed by DLS.

FIG. 9 shows the results of RP-HPLC analysis of a mixture of sevenfluorocarbon-linked peptides exposed to 50% (v/v) acetic acid for 24hours.

FIG. 10 shows photographs of formulated and unformulated mixtures offluorocarbon-linked peptides after being shaken by hand. Vial A:formulated+mannitol/water; Vial B: formulated+mannitol/histidine; VialC: non-formulated+mannitol/water; Vial D:non-formulated+mannitol/histidine.

FIG. 11 shows photographs of formulated and unformulated mixtures offluorocarbon-linked peptides after being vortexed and sonicated. Vial A:formulated+mannitol/water; Vial B: formulated+mannitol/histidine; VialC: non-formulated+mannitol/water; Vial D:non-formulated+mannitol/histidine.

FIG. 12 shows transmission electron micrographs of a formulationcomprising six fluorocarbon-linked influenza peptides (FP-01.1).

FIG. 13 shows the HPLC profile of post-filtered FR01.1 at t₀ (upperpanel) and after 24 hours (lower panel).

FIG. 14 shows the HPLC profile of FP01.1 after reconstitution in water.

FIG. 15 shows a comparison of reconstituted formulated fluorocarbonpeptides (FP01.1) and non-formulated peptides in water. The leftphotograph was taken after 20 minutes standing and the right photographwas taken after 3 minutes sonication.

FIG. 16 shows a comparison of reconstituted formulated fluorocarbonpeptides (FP01.1) and non-formulated peptides in 28 mM L-Histidine. Theleft photograph was taken after 20 minutes standing and the rightphotograph was taken after 3 minutes sonication.

FIG. 17 shows the volume adjusted dose response in rats. FP-01.1 induceda positive IFN-γ T cell response at all dose levels tested in a dosedependent fashion.

FIG. 18 shows vaccine-induced T cell responses observed using an ex vivoIFN-γ ELISpot assay. PBMCs were stimulated with 6 individual peptides(corresponding to peptides contained in the vaccine) for 18 hours.Positive assay responses were defined as the mean of number of spots inthe negative control wells+2 standard deviations of the mean. The numberof spots for each of the 6 peptides was cumulated to obtain the “sum forlong peptides” and expressed as a number of spots per million inputPBMCs.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The sequence listing corresponds to the peptides used in the Examples asshown in the Table below.

Sequence Peptide SEQ ID NO: 1 P1 without N-terminal lysine SEQ ID NO: 2P8 without N-terminal lysine SEQ ID NO: 3 P9 without N-terminal lysineSEQ ID NO: 4 P2 without N-terminal lysine SEQ ID NO: 5 P4 withoutN-terminal lysine SEQ ID NO: 6 P5 without N-terminal lysine SEQ ID NO: 7P1 variant without N-terminal lysine SEQ ID NO: 8 P8 variant withoutN-terminal lysine SEQ ID NO: 9 P8 variant without N-terminal lysine SEQID NO: 10 P10 without N-terminal lysine SEQ ID NO: 11 P3 withoutN-terminal lysine SEQ ID NO: 12 P4 variant without N-terminal lysine SEQID NO: 13 P6 without N-terminal lysine SEQ ID NO: 14 P7 withoutN-terminal lysine SEQ ID NO: 15 P11 without N-terminal lysine SEQ ID NO:16 P12 without N-terminal lysine SEQ ID NO: 17 P1 with N-terminal lysineSEQ ID NO: 18 P8 with N-terminal lysine SEQ ID NO: 19 P9 with N-terminallysine SEQ ID NO: 20 P2 with N-terminal lysine SEQ ID NO: 21 P4 withN-terminal lysine SEQ ID NO: 22 P5 with N-terminal lysine SEQ ID NO: 23P3 with N-terminal lysine SEQ ID NO: 24 P6 with N-terminal lysine SEQ IDNO: 25 P7 with N-terminal lysine SEQ ID NO: 26 P10 with N-terminallysine SEQ ID NO: 27 P11 with N-terminal lysine SEQ ID NO: 28 P12 withN-terminal lysine

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of formulatingfluorocarbon-linked peptides for administration to a human or animal andpharmaceutically acceptable fluorocarbon-linked peptide formulationsobtainable by the method of the invention. The method of the inventioncomprises the step of solubilising the fluorocarbon-linked peptide in anacidic solution, preferably in acetic acid. The invention also providesaqueous formulations which are acidic and so unsuitable foradministration to a human or animal but which are important forobtaining the pharmaceutically acceptable formulation.

Typically, at least one fluorocarbon-linked peptide used in theformulation process or present in the aqueous acidic formulation orpharmaceutically acceptable formulation of the invention comprises apeptide of at least 20 amino acid residues, having at least 50%hydrophobic amino acid residues and having an isoelectric point ofgreater than or equal to 7.

A formulation of the invention may comprise, or the formulation methodof the invention may use, at least one fluorocarbon-linked peptidewherein the peptide comprises at least about 20 amino acids in which atleast about 50% of the amino acids are hydrophobic. Otherfluorocarbon-linked peptides present in the formulation may be shorterthan 20 amino acids and/or may have fewer than 50% hydrophobic residues.

Formulations of the present invention may contain fluorocarbon-linkedpeptides comprising a sequence of at least seven amino acids up to about100 amino acids, such as from about 9 to about 50 amino acids,preferably from about 15 to about 45 amino acids, more preferably fromabout 20 to about 40 amino acids, such as from about 25 to about 38, forexample 30, 31, 32, 33, 34, 35, 36 or 37 amino acids.

The formulation of the invention may comprise at least onefluorocarbon-linked peptide, wherein at least 50% of the amino acids inthe peptide are hydrophobic. Typically, between about 50% and about 80%,such as about 70% or about 75%, of residues are hydrophobic. The lowerlimit could be 48% or 49%. Preferably, the peptide comprises from about55% to about 60 or about 65% hydrophobic residues. Where the formulationcomprises further fluorocarbon-linked peptides, the furtherfluorocarbon-linked peptides may have less than 50% hydrophobicity. Forexample, the peptide component of a further fluorocarbon-linked peptidemay comprise from about 30% to about 70% hydrophobic residues, forexample, about 40%, such as at about 45%, 50%, 55%, 60% or 65%hydrophobic residues. Tryptophan (W), tyrosine (Y), isoleucine (I),phenylalanine (F), leucine (L), valine (V), methionine (M), arginine(A), proline (P), glycine (G) and cysteine (C) are hydrophobic aminoacids. In a preferred embodiment, none of the peptides present in theformulation comprise a contiguous sequence of 20 or more amino acidresidues in which more than 80% of the residues are hydrophobic.

One or more of the further fluorocarbon-linked peptides may comprise apeptide that: is at least 20 amino acid residues long; comprises atleast 50% hydrophobic amino acid residues; and/or has an isoelectricpoint greater than or equal to 7. The first fluorocarbon-linked peptideand/or one or more of the further fluorocarbon-linked peptides maycomprise a peptide that: comprises a positively charged amino acid inthe last 15 contiguous amino acids distal to the fluorocarbon; and/ordoes not comprise a contiguous sequence of 20 amino acid residuescomprising more than 80% hydrophobic amino acid residues.

In one embodiment, none of the peptides in a formulation of theinvention has an isoelectric point of less than 7, does not comprise apositively charged amino acid in the last 15 contiguous amino acidsdistal to the fluorocarbon, and/or comprises a contiguous sequence of 20amino acid residues comprising more than 80% hydrophobic amino acidresidues.

The fluorocarbon-linked peptides in the formulation of the invention aretypically present in micelles with a diameter of less than 0.22 μm. Themicelles typically have a diameter of from about 15 to about 200 nm,typically from about 20 nm to about 100 nm, such as from about 20 nm toabout 30 nm or from about 30 nm to about 50 nm. However, some largermicelles may be present. In general, not more than 20%, such as fromabout 10% to about 15% of the aggregates have a diameter greater than100 nm. Preferably, at least 80% of the fluorocarbon-linked peptidemicelles have a diameter of less than 100 nm. Micelle size may bedetermined by any suitable method, such as by Dynamic Light Scattering(DLS) or using Transmission Electron Microscopy (TEM).

Formation of micelles may be facilitated by solubilising thefluorocarbon-linked peptides in an acidic solution. For example, thefluorocarbon-linked peptides may be solubilised in acetic acid asdescribed herein. The aqueous formulation of the invention for use inthe preparation of a pharmaceutically acceptable formulation may beacidic, having, for example, a pH of 5 or less.

The pharmaceutically acceptable formulation of the invention may be indried, such as lyophilized, form. The pharmaceutically acceptableformulation of the invention may be an aqueous solution, for example anformed by dissolving a lyophilisate or other dried formulation in anaqueous medium. The aqueous solution is typically pH neutral.

In an aqueous (liquid) formulation of the invention, the solution istypically clear with no visible aggregates. In particular, noparticulates are visible in the solution after perturbation by vortexingand sonication. This applies both to the acidic formulation and to thepharmaceutically acceptable formulation.

The peptide is typically a peptide antigen or allergen capable ofinducing an immune response in an animal, including humans, i.e. thepeptide is typically an immunogenic peptide. Preferably the immuneresponse will have a beneficial effect in the host. Immunogenic peptidesmay be derived from an infectious agent (pathogen), such as a virus,bacterium, Mycobacterium, parasite or fungus or from an autologousprotein, such as a cancer antigen (protein derived from a tumour cell),or from an allergen.

Examples of viruses include and are not limited to animal and humanviruses such as: influenza, Human Immunodeficiency Virus (HIV),Hepatitis C Virus (HCV), Hepatitis B Virus (HBV), Hepatitis A Virus(HAV), Respiratory Syncytial Virus (RSV), Venezuelan Equine Encephalitisvirus (VEE), Japanese Encephalitis virus (JEV), Cytomegalovirus (CMV),Epstein Barr Virus (EBV), Herpes Virus (HSV-1 or HSV-2), Ebola, Marburg,Dengue, West Nile and Yellow fever viruses, Porcine reproductive andrespiratory syndrome virus (PRRSV) and Feline Immunodeficiency Virus(FIV).

Examples of bacteria and mycobacteria include, but are not limited toMycobacterium tuberculosis, Legionella, Rickettsiae, Chlamydiae, andListeria monocytogenes.

Examples of parasites include, but are not limited to Plasmodiumfalciparum and other species of the Plasmodial family.

Examples of fungi include, but are not limited to Candida albicans,Cryptococcus, Rhodotorula and Pneumocystis.

Autologous or self-antigens include, but are not limited to thefollowing antigens associated with cancers, P53, MAGE-A3, NY-ESO-1,SURVIVIN, WT1, HER-2/neu, MUC 1, hTERT, MAGE-1, LAGE-1, PAP, T21, TRP-2,PSA, Livin, HAGE, SSX-1, PRAME, PASD1, IMP-3, SSX-4, CDCA-1 and/or BAGE.

Allergens include, but are not limited to. phospholipase A₂ (API ml)associated with severe reactions to bee, Derp-2, Der p 2, Der f, Der p 5and Der p 7 associated with reaction against the house-dust miteDermatophagoides pteronyssinus, the cockroach allergen Bla g 2 and themajor birch pollen allergen Bet v 1.

In one embodiment, the peptide is derived from the influenza virus. Theinfluenza peptide antigen may comprise one or more epitopes from aninfluenza type A protein, an influenza type B protein or an influenzatype C protein. Examples of the influenza virus proteins, from both theinfluenza A and B types, include: haemagglutinin, neuraminidase, matrix(M1) protein, M2, nucleoprotein (NP), PA, PB1, PB2, NS1 or NS2 in anysuch combination.

As used herein the term “immunogenic” refers to a molecule having theability to be recognised by immunological receptors such as T cellreceptor (TCR) or B cell receptor (BCR or antibody). The immunogenicpeptide may be natural or non-natural, provided it presents at least oneepitope, for example a T cell and/or a B cell epitope. The peptide maycontain one or more T cell epitopes, including T helper cell epitopesand/or cytotoxic T lymphocyte (CTL) epitopes, and/or one or more B cellepitopes or combinations of T and B cell epitopes, such as MHC class Ior MHC class II epitopes. Methods for identifying epitopes are wellknown in the art.

The peptide may comprise one or more epitopes. The peptide may comprisemore than one epitope linked together. One such example is the use offusion peptides where a promiscuous T helper epitope can be covalentlylinked to one or multiple CTL epitopes or one or multiple B cellepitope. As an example, the promiscuous T helper epitope could be thePADRE peptide, tetanus toxoid peptide (830-843) or influenzahaemagglutinin, HA(307-319).

The epitopes may be overlapping linear epitopes so that the peptidecomprises a cluster of densely packed multi-specific epitopes.

The terminus of the peptide that is not conjugated to the fluorocarbonattachment may be altered to promote solubility of the construct via theformation of micelles. For example, a positively charged amino acidcould be added to the peptide in order to promote the assembly ofmicelles. Either the N-terminus or the C-terminus of the peptide may becoupled to the vector to create the construct. To facilitate large-scalesynthesis of the construct, the N- or C-terminal amino acid residues ofthe peptide can be modified. When the desired peptide is particularlysensitive to cleavage by peptidases, the normal peptide bond can bereplaced by a non-cleavable peptide mimetic. Such bonds and methods ofsynthesis are well known in the art.

Non-standard, non-natural amino acids can also be incorporated inpeptide sequences provided that they do not interfere with the abilityof the peptide to interact with MHC molecules and remain cross-reactivewith T cells recognising the natural sequences. Non-natural amino acidscan be used to improve peptide resistance to protease or chemicalstability. Examples of non-natural amino acids include the D-amino acidsand cysteine modifications.

The peptide may be derived by purification from the native protein orproduced by recombinant technology or by chemical synthesis. Methods forthe preparation of peptides are well known in the art.

It will be understood by vaccine designers that more than one peptidemay be required to provide a broader prophylactic or immunotherapeuticeffect. Such multi-component products are desirable since they arelikely to be more effective at eliciting appropriate immune responses.For example, the optimal formulation of an influenza vaccine maycomprise a number of peptide epitopes from different influenza proteinsor the optimal formulation of an HIV immunotherapeutic may comprise anumber of epitopes from different HIV proteins. Alternatively, multipleepitopes may be incorporated into a formulation in order to conferimmunity against a range of pathogens. For example a respiratoryinfection vaccine may contain epitopes from influenza virus andrespiratory syncytial virus.

A formulation of the invention may comprise multiple immunogenicpeptides. Typically each peptide comprises a different epitope. Eachpeptide may be linked to a common fluorocarbon vector. More practically,combinations of fluorocarbon-linked peptides may be present in aformulation of the invention, wherein different peptides areindependently linked to fluorocarbon chains. In a mixture offluorocarbon-linked peptides, each peptide may be linked to afluorocarbon chain of a single structure. Alternatively, the mixture maycomprise peptides linked to fluorocarbon chains with differentstructures.

A formulation of the invention may comprise one or morefluorocarbon-linked peptides, preferably from about 2 to about 20,preferably about 3 to about 10. In particular embodiments the multicomponent vaccine may contain 4, 5, 6, 7, 8 or 9 fluorocarbon-linkedpeptides. This aids the generation of a multi-epitopic immune response.

The different peptides present in a multi-component product may bedifferent antigens from the same pathogen, or may be antigens fromdifferent pathogens. Alternatively, the peptides may be different tumorantigens or antigens from different parts of an autologous protein.

Fluorocarbon-linked peptides comprising immunogenic influenza peptidesare used in the Examples. The present invention is not limited to theseparticular peptides but extends to any immunogenic peptides having theproperties described above. However, preferred formulations of theinvention include one or more of the following six immunogenic influenzapeptides that are selected from highly conserved segments of the PA,PB1, PB2, NP & M1 proteins:

(SEQ ID NO: 1) HMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADK (SEQ ID NO: 2)VAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQG (SEQ ID NO: 3)YITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFE (SEQ ID NO: 4)APIMFSNKMARLGKGYMFESKRMKLRTQIPAEMLA (SEQ ID NO: 5)DQVRESRNPGNAEIEDLIFLARSALILRGSVAHKS (SEQ ID NO: 6)DLEALMEWLKTRPILSPLTKGILGFVFTLTVPSER

The peptides are preferably each separately linked to a fluorocarbonvector. Particularly preferred formulations of the invention compriseall six of the above fluorocarbon-linked peptides and do not includefluorocarbon-linked peptides comprising peptides having the sequencesshown in any one of SEQ ID NOs: 13, 14, 23 and 24. Otherfluorocarbon-linked peptides may be included in the preferredformulations of the invention. However, it is preferred that theformulation comprises the six fluorocarbon-linked peptides describedabove and no other fluorocarbon-linked peptides.

One or more of the six peptides may be substituted by a variant peptidecomprising one, two or three amino acid substitutions. The variantpeptides may comprises a sequence derived from different influenzastrains. For example, SEQ ID NO: 1 may be replaced by SEQ ID NO: 7, SEQID NO: 2 may be replaced by SEQ ID NO: 8 or 9, SEQ ID NO: 3 may bereplaced by SEQ ID NO: 10, SEQ ID NO: 4 by SEQ ID NO: 11 and/or SEQ IDNO: 5 by SEQ ID NO: 12.

The peptides may be linked to the fluorocarbon vector via a spacermoiety as described below. The spacer moiety is preferably a lysineresidue. Accordingly, the preferred formulation of the invention maycomprise fluorocarbon-linked peptides in which the peptides have one ormore of the sequences shown in SEQ ID NOs: 17 to 22. The N-terminallysine in the peptides is preferably linked to a fluorocarbon having theformula C₈F₁₇ (CH₂)₂COOH. The fluorocarbon is preferably coupled to theepsilon chain of the N-terminal lysine residue.

Thus, in one preferred embodiment, the invention provides apharmaceutically acceptable formulation consisting of, or consistingessentially of, six fluorocarbon-linked peptides comprising SEQ ID NOs:1 to 6 and a pharmaceutically acceptable carrier or diluent andoptionally an adjuvant.

In each of the six fluorocarbon-linked peptides, the peptides preferablyconsist of one of SEQ ID NOs: 1 to 6 with an N-terminal lysine residueadded (i.e. one of SEQ ID NOs: 17 to 22), which lysine residue iscoupled to a fluorocarbon chain having the formula C₈F₁₇ (CH₂)₂COOH viathe epsilon chain of the lysine residue.

The fluorocarbon attachment in the fluorocarbon-linked peptide maycomprise one or more chains derived from perfluorocarbon or mixedfluorocarbon/hydrocarbon radicals, and may be saturated or unsaturated,each chain having from 3 to 30 carbon atoms.

Thus, the chains in the fluorocarbon attachment are typically saturatedor unsaturated, preferably saturated. The chains in the fluorocarbonattachment may be linear or branched, but are preferably linear. Eachchain typically has from 3 to 30 carbon atoms, preferably from 5 to 25,more preferably from 8 to 20.

In order to covalently link the fluorocarbon attachment to the peptide,a reactive group, or ligand, for example —CO—, —NH—, S, O or any othersuitable group is included. The use of such ligands for achievingcovalent linkages is well known in the art. The reactive group may belocated at any position on the fluorocarbon molecule.

Coupling of the fluorocarbon moiety to the peptide may be achievedthrough functional groups such as —OH, —SH, —COOH and —NH₂ naturallypresent or introduced onto any site of the peptide. Examples of suchlinkages include amide, hydrazone, disulphide, thioether and oximebonds.

Optionally, a spacer element (peptidic or non-peptidic) may beincorporated to permit cleavage of the peptide from the fluorocarbonelement for processing within an antigen-presenting cell and to optimisesteric presentation of the peptide. The spacer may also be incorporatedto assist in the synthesis of the molecule and to improve its stabilityand/or solubility. Examples of spacers include polyethylene glycol (PEG)or amino acids such as lysine or arginine that may be cleaved byproteolytic enzymes.

In one embodiment, the fluorocarbon-linked peptide may have the chemicalstructure C_(m)F_(n)—C_(y)H_(x)—(Sp)-R or derivatives thereof, where m=3to 30, n≤2m+1, y=0 to 15, x≤2y, (m+y)=3 to 30 and Sp is an optionalchemical spacer moiety and R is a peptide antigen. Typically m and nsatisfy the relationship 2m−1≤n≤2m+1, and preferably n=2m+1. Typically xand y satisfy the relationship 2y-2≤x≤2y, and preferably x=2y.Preferably the C_(m)F_(n)—C_(y)H_(x) moiety is linear.

It is preferred that m is from 5 to 15, more preferably from 8 to 12. Itis also preferred that y is from 0 to 8, more preferably from 0 to 6 or0 to 4. It is therefore particularly preferred that theC_(m)F_(n)—C_(y)H_(x) moiety is saturated (i.e. n=2m+1 and x=2y) andlinear, and that m=8 to 12 and y=0 to 6 or 0 to 4.

In a particular example, the fluorocarbon attachment is derived from 2H,2H, 3H, 3H-perfluoroundecanoic acid of the following formula:

Thus, a preferred fluorocarbon attachment is the linear saturated moietyC₈F₁₇(CH₂)₂—. Further examples of fluorocarbon attachments have thefollowing formulae: C₆F₁₃(CH₂)₂—, C₇F₁₅(CH₂)₂—, C₉F₁₉(CH₂)₂—,C₁₀F₂₁(CH₂)₂—, C₅F₁₁(CH₂)₃—, C₆F₁₃(CH₂)₃—, C₇F₁₅(CH₂)₃—, C₈F₁₇(CH₂)₃—and C₉F₁₉(CH₂)₃— which are derived from C₆F₁₃(CH₂)₂COOH,C₇F₁₅(CH₂)₂COOH, C₉F₁₉(CH₂)₂COOH, C₁₀F₂₁(CH₂)₂COOH, C₅F₁₁(CH₂)₃COOH,C₆F₁₃(CH₂)₃COOH, C₇F₁₅(CH₂)₃COOH, C₈F₁₇(CH₂)₃COOH and C₉F₁₉(CH₂)₃COOHrespectively.

Preferred examples of suitable structures for the fluorocarbonvector-antigen constructs have the formula:

in which Sp and R are as defined above. Preferably Sp is derived from alysine residue and has the formula —CONH—(CH₂)₄—CH(NH₂)—CO—. PreferablyR is any one of SEQ ID NOs: 1 to 6. The amino group of the N-terminalamino acid of SEQ ID NO: 1, 2, 3, 4, 5 or 6 thus forms an amide linkagewith the C-terminal carboxy group of the spacer of formula—CONH—(CH₂)₄—CH(NH₂)—CO—.

In the context of the current invention the fluorocarbon attachment maybe modified such that the resulting compound is still capable ofdelivering the peptide to antigen presenting cells. Thus, for example, anumber of the fluorine atoms may be replaced with other halogen atomssuch as chlorine, bromine or iodine. In addition, it is possible toreplace a number of the fluorine atoms with methyl groups and stillretain the properties of the molecule described herein.

The present invention provides both an acidic aqueous formulation and apharmaceutically acceptable formulation comprising one or more, such astwo or more, fluorocarbon-linked peptides and optionally apharmaceutically acceptable carrier or excipient. Preferably, at leastone fluorocarbon-linked peptide in the formulation comprises a peptideof at least 20 amino acid residues, having at least 50% hydrophobicamino acid residues and having an isoelectric point of greater than orequal to 7. The excipient may be a stabilizer or bulking agent necessaryfor efficient lyophilisation. Examples include sorbitol, mannitol,polyvinylpyrrolidone and mixtures thereof, preferably mannitol. Otherexcipients that may be present include preservatives such asantioxidants, lubricants, cryopreservatives and binders well known inthe art.

The present invention provides a method for preparing the formulationsof the invention.

In a method of the invention, at least one fluorocarbon-linked peptideis solubilised in acetic acid as a first step in formulating apharmaceutical product. The fluorocarbon-linked peptide used as astarting material is typically in desiccated form. Thefluorocarbon-linked peptide(s) solubilised in acetic acid typicallycomprises a peptide that is at least about 20 amino acids long in whichat least about 50% of the amino acids are hydrophobic. Otherfluorocarbon-linked peptides may be solubilised in acetic acid or inother solvents. In particular, fluorocarbon-linked peptides thatcomprise peptides shorter than 20 amino acids and/or that have fewerthan 50% hydrophobic residues may be solubilised in a solvent other thanacetic acid.

The term “solubilisation” is used herein to mean the dispersion offluorocarbon-linked peptides in a solvent to form a visually clearsolution that does not lose material upon sterile filtration. Thefluorocarbon-linked peptides may be present in a multimolecular micellarstructure. By “dispersion” is meant dissolution of the lyophilizedfluorocarbon-linked peptides in order to disrupt particulates andachieve solubility through the formation of micellar structures.

The term “aggregates” is used herein to describe macromolecularfluorocarbon-linked peptide structures. Micellar aggregates offluorocarbon-linked peptides may assist in solubilisation. Grossaggregates of fluorocarbon-linked peptides result in visibleparticulates. The term “particulates” is used herein to mean aggregatesof fluorocarbon-linked peptides visible to the naked eye.

Solubilisation of the fluorocarbon-linked peptide in acetic acidtypically results in the formation of a clear solution containingmicellar aggregates of fluorocarbon-linked peptides. The micellaraggregates typically have a diameter of from about 20 nm to about 50 nm,for example from about 17 nm to about 30 nm. However, some largeraggregates having a diameter of more than about 50 nm, typically notmore than 20%, such as from about 10 to about 15% of the aggregates havea diameter greater than 100 nm. Preferably, no aggregates are visible tothe naked eye. Particle size may be determined by any suitable method,such as by Dynamic Light Scattering (DLS) or using Transmission ElectronMicroscopy (TEM). For example, using TEM in negative staining, 20 μl offluorocarbon-linked peptide solution is deposited on a Formvar carboncoated copper electron microscope grid (300 mesh). 20 μl of uranyleacetate (1% aqueous) is then added. After 30 seconds, excess solution isquickly wicked away with a Whatman filter paper. The sample is thenallowed to dry for at least 2 minutes before analysis. Transmissionelectron microscopy is then performed on Philips CM120 biotwin at 120 kVaccelerating voltage. Image acquisition is performed at a directmagnification ranging from 50000× to 150000×.

The concentration of fluorocarbon-linked peptide in the solution istypically from about 0.1 mM to about 10 mM, such as about 0.5 mM, 1 mM,2 mM, 2.5 mM or 5 mM. An example of a suitable concentration is about 10mg/ml.

Examples of typical manufacturing process flows, commencing at theinitial solubilisation step through to final product presentation areprovided in FIGS. 1 and 2. These emphasise the requirement for thesolvent to not only achieve fluorocarbon-linked peptide solubility butalso for it to be compatible with downstream processes includingblending with potential stabilizers, sterile filtration andlyophilisation. In the flowcharts the letter n is used to denote avariable number of additional fluorocarbon-linked peptides that could beincluded in the formulation.

Variations to the process flow are permitted, as known to one skilled inthe art, to achieve the same resulting product characteristics; namely,that the input components are blended homogenously together to thedesired ratios with any aggregates dispersed, rendered sterile andpresented in a suitable format for administration. Such examples couldinclude the introduction of a vortexing and/or sonication post-blendingor post-dilution stage to facilitate solubilisation. Other permutationsof the manufacturing process flow could include sterile filtration beingperformed at an earlier stage of the process or the omission oflyophilisation to permit a liquid final presentation.

Alternatively, one set of fluorocarbon-linked peptides may besolubilised individually in one organic solvent, then blended togetherand sterile filtered, with a second set of fluorocarbon-linked peptidesbeing solubilised in an alternative solvent, blended and sterilefiltered (FIG. 2) before the two sets of fluorocarbon-linked peptidesare blended together for further processing.

The initial solvent may be the same or different for eachfluorocarbon-linked peptide so that one of more of thefluorocarbon-linked peptides may be solubilised in acetic acid and oneor more of the fluorocarbon-linked peptides may be solubilised inanother solvent having acceptable properties. For example in the ProcessFlow of FIG. 1, B may be a different solvent to A.

Alternatively, acetic acid may be used as the initial solvent fordifferent fluorocarbon-linked peptides, but may be used at different,optimised, concentrations for the different fluorocarbon-linkedpeptides. For example in the Process Flow of FIG. 1, B may be adifferent concentration of the same solvent as A.

The different fluorocarbon-linked peptides may be mixed prior tosolulilisation.

The acetic acid may be used at a concentration of from 5 to 80% (v/v)aqueous acetic acid, such as at a concentration of from 10 to 70% (v/v),such as a concentration of about 20% (v/v) or 50% (v/v). In a method forformulating a mixture of fluorocarbon-linked peptides, differentpeptides may be solubilised in different concentrations of acetic acidprior to blending. For example, one or more fluorocarbon-linked peptidemay be solubilised in 10% (v/v) acetic acid and one or more peptide maybe solublised in 80% (v/v) acetic acid.

Where more than one solvent is used in the manufacturing process, eachsolvent used is typically: able to solubilise the fluorocarbon-linkedpeptide it is being used to solubilise at relatively high concentrations(for example, up to 10 millimolar, such as up to 2 millimolar);water-miscible to facilitate dilution with water prior tolyophilisation; compatible with lyophilisation stabilizers, such asmannitol, that may be used in the manufacturing process; has a safetyprofile acceptable to the pharmaceutical regulatory authorities, forexample, complies with the requirements of ICH Q3C (Note for Guidance onImpurities: Residual Solvents) and the requirements of Class IIIsolvents, as defined by USP Residual Solvents <467> (residual solventlimit of 50 mg/day in finished product or less than 5000 ppm or 0.5%);amenable to lyophilisation, that is, sufficiently volatile to be removedto safe levels upon lyophilisation; able to disperse thefluorocarbon-linked peptide molecules efficiently in a reproducible anduniform manner such that yield losses on sterilising grade filtrationare minimised; unable to react with, or promote degradation of, thefluorocarbon-linked peptide molecule; and/or compatible with thematerials routinely used in pharmaceutical product manufacture(containers/filter membranes/pipework etc).

Examples of solvents that may be used to disperse one or more of thefluorocarbon-linked peptides in the blend include phosphate bufferedsaline (PBS), propan-2-ol, tert-butanol, acetone and other organicsolvents.

Where the different fluorocarbon-linked peptides are solubilisedseparately, for example in different solvents or in differentconcentrations of acetic acid, the solubilised peptides are blended tocreate a mixture of fluorocarbon-linked peptides.

One or more pharmaceutically acceptable excipients and/or adjuvants mayalso be added to the solubilised fluorocarbon-linked peptide or mixtureof fluorocarbon-linked peptides.

By “excipient” is meant an inactive substance used as a carrier for thefluorocarbon-linked peptides. Typically, the solubilisedfluorocarbon-linked peptides are mixed with the excipient. Potentialexcipients that may be used in the manufacturing process includestabilizers or bulking agents necessary for efficient lyophilisation.Examples include sorbitol, mannitol, polyvinylpyrrolidone and mixturesthereof, preferably mannitol. Other excipients include preservativessuch as antioxidants, lubricants, cryopreservatives and binders wellknown in the art.

To enhance the breadth and intensity of the immune response mounted tothe peptide antigen, one or more adjuvant and/or otherimmuno-potentiating agent may be included in the formulation. An“adjuvant” in this context is an agent that is able to modulate theimmune response directed to a co-administered antigen while having fewif any direct effects when given on its own. Such adjuvants may becapable of potentiating the immune response in terms of magnitude and/orcytokine profile.

Suitable adjuvants include:

(1) natural or synthetically derived refinements of natural componentsof bacteria such as Freund's adjuvant & its derivatives,muramyldipeptide (MDP) derivatives, CpG, monophosphoryl lipid A;

(2) other known adjuvant or potentiating agents such as saponins,aluminium salts and cytokines;

(3) oil in water adjuvants, such as the submicron oil-in water emulsionMF-59, water-in-oil adjuvants, immunostimulating complex (ISCOMs),liposomes, formulated nano- and micro-particles;

(4) bacterial toxins and toxoids; and

(5) other useful adjuvants well known to one skilled in the art.

After solubilisation and blending the solution of fluorocarbon-linkedpeptide(s) is diluted. For example, the blend may be diluted in water.

The solution containing the fluorocarbon-linked peptides is preferablysterilised. Sterilisation is particularly preferred where theformulation is intended for systemic use. Any suitable means ofsterilisation may be used, such as UV sterilisation or filtersterilisation. Preferably, filter sterilisation is used. Sterilefiltration may include a 0.45 μm filter followed by a 0.22 μmsterilizing grade filter train.

Sterilisation may be carried out before or after addition of anyexcipients and/or adjuvants.

After filter sterilisation, the yield of the fluorocarbon-linked peptidepresent in the sterile solution is typically at least 80%, preferably atleast 90%, more preferably at least 95% of the amount offluorocarbon-linked peptide present before sterilisation. A yield ofmore than 95%, such as a yield of 98%, 99% or more, such as a yield of100%, may be achieved.

After sterilisation, the fluorocarbon-linked peptide is typicallypresent in the solution in micellar structures having diameters of fromabout 20 nm to about 100 nm, such as about 30 nm or about 50 nm. Largerparticles present in the solution prior to sterilisation may typicallybe reshaped by filter sterilisation. The sterilized solution may bestored in a sterile container.

The sterile formulation is dried to remove the acetic acid. Drying theformulation also facilitates long-term storage. Any suitable dryingmethod may be used. Lyophilisation is preferred but other suitabledrying methods may be used, such as vacuum drying, spray-drying, sprayfreeze-drying or fluid bed drying. The drying procedure can result inthe formation of an amorphous cake within which the fluorocarbon-linkedpeptides are incorporated.

For long-term storage, the sterile formulation may be lyophilized.Lyophilisation can be achieved by freeze-drying. Freeze-drying typicallyincludes freezing and then drying. For example, the fluorocarbon-linkedpeptide mixture may be frozen for 2 hours at −80° C. and freeze-dried ina freeze drying machine for 24 hours.

Pharmaceutically acceptable formulations of the invention may be solidcompositions. The fluorocarbon-linked peptide composition may beobtained in a dry powder form. A cake resulting from lyophilisation canbe milled into powder form. A solid composition according to theinvention thus may take the form of free-flowing particles. The solidcomposition is typically provided as a powder in a sealed vial, ampouleor syringe. If for inhalation, the powder can be provided in a drypowder inhaler. The solid matrix can alternatively be provided as apatch. A powder may be compressed into tablet form.

The dried, for example lyophilized, fluorocarbon-linked peptideformulation may be reconstituted prior to administration. The term“reconstitution” as used herein means dissolution of the dried vaccineproduct prior to use. Following drying, such as lyophilisation, thefluorocarbon-linked peptide product is preferably reconstituted to forman isotonic, pH neutral, homogeneous suspension. The formulation istypically reconstituted in the aqueous phase, for example by addingWater for Injection (Hyclone), histidine buffer solution (such as 28 mML-histidine buffer) or phosphate buffered saline (PBS). Thereconstituted formulation is typically dispensed into sterilecontainers, such as vials, syringes or any other suitable format forstorage or administration.

The invention provides a fluorocarbon-linked peptide formulationobtainable by a method according to the invention. The formulation maybe an intermediate in the preparation of a pharmaceutical product.

The invention provides an aqueous formulation suitable for use as anintermediate in the preparation of a fluorocarbon-linked peptideformulation for administration to a human or animal, which aqueouscomposition is acidic and comprises one or more solubilisedfluorocarbon-linked peptide as described above. The acidic solutiontypically comprises acetic acid. At least one of the fluorocarbon-linkedpeptides may be at least 20 amino acid residues long, comprise at least50% hydrophobic amino acid residues and have an isoelectric pointgreater than or equal to 7; and/or be present in micelles with adiameter of less than 0.22 μm. The aqueous solution is preferablysterile. It may further comprise a pharmaceutically acceptable carrieror diluent.

The invention also provides a pharmaceutically acceptablefluorocarbon-linked peptide formulation. The pharmaceutically acceptableformulation may be a solid, such as a powder, cake or tablet. Thepharmaceutical formulation may be an aqueous solution.

The formulation may be stored in a container, such as a sterile vial orsyringe.

The invention thus provides, in one embodiment, a formulation comprisinga fluorocarbon-linked peptide, wherein the fluorocarbon-linked peptideis present in micellar structures, and a pharmaceutically acceptableamount of acetic acid. In other formulations of the invention, theacetic acid is completely removed by the drying step.

The ICH recommended maximum of acetic acid is 50 mg per day. Typically,the acetate level in a formulation of the invention is less than 5000ppm or 0.5% in accordance with the requirements for Class III solventsdefined by USP Residual Solvents <467>. The invention also provides anintermediate formulation comprising a fluorocarbon-linked peptidesolubilised in acetic acid.

In one aspect, the present invention provides a formulation comprisingtwo or more fluorocarbon-linked peptides, wherein thefluorocarbon-linked peptides are present in micellar structures.

In another aspect, the invention provides a formulation comprising afluorocarbon-linked peptide, wherein the fluorocarbon-linked peptide ispresent in micellar structures and the formulation is in lyophilisedform.

In a formulation of the invention, the fluorocarbon-linked peptides aretypically present in multiple micellar structures. The micellarstructures typically have a diameter of from about 20 nm to about 100nm, such as about 50 nm or 70 nm. It is preferred than at least 80%,such as at least 90% or at least 95% of the micellar structures presentin the formulation have a diameter of less than 100 nm such as adiameter of from about 20 nm to about 50 nm.

In a further aspect, the formulation of the present invention furthercomprises a pharmaceutically acceptable excipient and/or adjuvant. Forexample, in one embodiment the formulation further comprises mannitoland/or other excipients.

In another aspect the invention provides the use of the formulation ofthe invention in the manufacture of a medicament for inducing an immuneresponse in a human or animal. The invention also provides the use ofthe formulation of the invention in the manufacture of a medicament fortreating or preventing of a disease of the human or animal body.

In a further aspect, the invention provides the formulation of theinvention for use in a method of treating the human or animal body bytherapy. Also provided in the formulation of the invention for use in amethod of stimulating an immune response in a human or animal and theformulation of the invention for use in a method of for treating orpreventing of a disease of the human or animal body.

In a further aspect, the invention provides a method of inducing animmune response in a human or animal in need thereof, said methodcomprising administering to said human or animal a prophylactic ortherapeutic amount of a formulation of the present invention. The immuneresponse may be effective in the treatment or prevention of a disease.

The disease is typically an infectious disease, an autoimmune disease,an allergy, a hormonal disease or cancer. The fluorocarbon-linkedpeptide in the formulation is selected to include one or more epitopesfrom the pathogen causing the infectious disease, the autologous proteinimplicated in the autoimmune disease or hormonal disease, the allergenresponsible for the allergy or a tumor antigen expressed on the cancercells.

Examples of infectious diseases that may be treated or prevented using afluorocarbon-linked peptide formulation of the invention include, butare not restricted to, infections caused by the following viruses,bacteria, mycobacteria, parasites and fungi: influenza, HumanImmunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Hepatitis B Virus(HBV), Hepatitis A Virus (HAV), Respiratory Syncytial Virus (RSV),Venezuelan Equine Encephalitis virus (VEE), Japanese Encephalitis virus(JEV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Herpes Virus(HSV-1 or HSV-2), Ebola, Marburg, Dengue, West Nile and Yellow feverviruses, Porcine reproductive and respiratory syndrome virus (PRRSV),Feline Immunodeficiency Virus (FIV), Mycobacterium tuberculosis,Legionella, Rickettsiae, Chlamydiae, and Listeria monocytogenes,Plasmodium falciparum and other species of the Plasmodial family,Candida albicans, Cryptococcus, Clostridium tetani, Rhodotorula andPneumocystis.

Examples of cancers that may be treated or prevented using afluorocarbon-linked peptide formulation of the invention include breastcancer, melanoma, colorectal cancer nasopharyngeal carcinoma, Burkitt'slymphoma and other human cancers.

In a preferred embodiment the formulation of the invention is used totreat or vaccinate against influenza. In a further aspect of thisembodiment, the influenza vaccine formulation may be administered incombination with an anti-viral therapeutic composition, includingneuraminidase inhibitor treatments such as amanidine, rimantidine,zanamivir or oseltamivir. In a still further aspect, the influenzavaccine formulation may be administered in combination with otherinfluenza vaccines, such as conventional antibody generating influenzavaccines. The other influenza vaccine is preferably a seasonal influenzavaccine.

Administration may be contemporaneous or separated by time. Thepharmaceutically acceptable formulation of the invention may beadministered before, together with or after the anti-viral therapeuticcomposition and/or other influenza vaccine.

The formulations comprising influenza peptides, in particular the sixinfluenza peptides having the sequences shown in SEQ ID NOs: 1 to 6, areprovided for use in a method of vaccinating against influenza.Accordingly, pharmaceutically acceptable formulations of the inventioncomprising such peptide may be used in the manufacture of a medicamentfor treating or preventing influenza. The invention also provides amethod of treating or preventing influenza, which method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of the fluorocarbon-linked influenza peptide formulations of theinvention.

Formulations of the invention may be administered to a human or animalsubject in vivo using a variety of known routes and techniques. Forexample, the formulation may be provided as an injectable solution,suspension or emulsion and administered via parenteral, subcutaneous,oral, epidermal, intradermal, intramuscular, interarterial,intraperitoneal, intravenous injection using a conventional needle andsyringe, or using a liquid jet injection system. The formulation may beadministered topically to skin or mucosal tissue, such as nasally,intratrachealy, intestinally, sublingually, rectally or vaginally, orprovided as a finely divided spray suitable for respiratory or pulmonaryadministration.

In one embodiment, the method of the invention further comprises thestep of processing the mixture into a formulation suitable foradministration as a liquid injection. Preferably, the method furthercomprises the step of processing the mixture into a formulation suitablefor administration via ingestion or via the pulmonary route.

The formulation is administered to a subject in an amount that iscompatible with the dosage formulation and that will be prophylacticallyand/or therapeutically effective. The administration of the formulationof the invention may be for either “prophylactic” or “therapeutic”purpose. As used herein, the term “therapeutic” or “treatment” includesany one or more of the following: the prevention of infection orreinfection; the reduction or elimination of symptoms; and the reductionor complete elimination of a pathogen. Treatment may be effectedprophylactically (prior to infection) or therapeutically (followinginfection).

The choice of carrier if required is frequently a function of the routeof delivery of the composition. Within this invention, compositions maybe formulated for any suitable route and means of administration.Pharmaceutically acceptable carriers or diluents include those used informulations suitable for oral, ocular, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, transdermal)administration.

The formulation may be administered in any suitable form, for example asa liquid, solid, aerosol, or gas. For example, oral formulations maytake the form of emulsions, syrups or solutions or tablets or capsules,which may be enterically coated to protect the active component fromdegradation in the stomach. Nasal formulations may be sprays orsolutions. Transdermal formulations may be adapted for their particulardelivery system and may comprise patches. Formulations for injection maybe solutions or suspensions in distilled water or anotherpharmaceutically acceptable solvent or suspending agent.

The appropriate dosage of the vaccine or immunotherapeutic to beadministered to a patient will be determined in the clinic. However, asa guide, a suitable human dose, which may be dependent upon thepreferred route of administration, may be from 1 to 1000 μg, such asabout 100 μg, 200 μg or 500 μg. Multiple doses may be required toachieve an immunological or clinical effect, which, if required, will betypically administered between 2 to 12 weeks apart. Where boosting ofthe immune response over longer periods is required, repeat doses 1month to 5 years apart may be applied.

The following Examples illustrate the invention.

Example 1: Synthesis of Peptides

Peptides having the amino acid sequences shown in SEQ ID NOs: 1 to 6,10, 11 and 13 to 16 were synthesised. The synthesis of each peptide wasperformed on solid phase using a classical Fmoc/t-butyl strategy and aTentaGel HL NH2 resin. A lysine residue was added to the N-terminus ofeach sequence. The sequences with the N-terminal lysine residue addedare shown in SEQ ID NOs: 17 to 28. After the addition of an N-terminalLysinyl residue, the resin block was split into two parts. One part wasused to incorporate the fluorocarbon chain (C₈F₁₇(CH₂)₂COOH) on theepsilon-chain of the N-terminal lysine to derive the fluorocarbon-linkedpeptide (FCP). With the second part, acetylation of the epsilon-chain ofthe N-terminal lysine was performed to derive the native peptide for usein comparative studies. Purified fluorocarbon-linked peptide (FCP) andnative peptides were obtained through cleavage in the presence oftrifluoroacetic acid (TFA) and a final purification by reversephase-high performance liquid chromatography (RP-HPLC). Both the FCPsand the native peptides described below possess an amido-group at theC-terminus. All preparations had a purity of 95% or greater and werepresented a dry, lyophilised powder. Net peptide mass was calculatedbased on nitrogen content analysis.

The following peptides P1 to P12 were linked to a fluorocarbon chain tocreate the FCPs, or were acetylated to create the native peptides (thestandard single letter code representation of amino acids has been used;X=Fluorocarbon vector (fluorocarbon-linked peptides); Z=acetyl (nativepeptides)):

P1: (SEQ ID NO: 1) NH2-K(X or Z)HMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADK-CONH₂ P2: (SEQ ID NO: 4)NH2-K(X or Z)APIMFSNKMARLGKGYMFESKRMKLRTQIPAEMLA- CONH₂ P3:(SEQ ID NO: 11) NH2-K(X or Z)APIMFSNKMARLGKGYMFESKSMKLRTQIPAEMLA- CONH₂P4: (SEQ ID NO: 5) NH2-K(X or Z)DQVRESRNPGNAEIEDLIFLARSALILRGSVAHKS-CONH₂ P5: (SEQ ID NO: 6)NH2-K(X or Z)DLEALMEWLKTRPILSPLTKGILGFVFTLTVPSER- CONH₂ P6:(SEQ ID NO: 13) NH2-K(X or Z)SPGMMMGMFNMLSTVLGVSILNLGQKKYTKTTY- CONH₂P7: (SEQ ID NO: 14) NH2-K(X or Z)KKKSYINKTGTFEFTSFFYRYGFVANFSMELPSFG-CONH₂ P8: (SEQ ID NO: 2)NH2-K(X or Z)VAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQG- CONH₂ P9:(SEQ ID NO: 3) NH2-K(X or Z)YITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFE- CONH₂P10: (SEQ ID NO: 10) NH2-K(X or Z)YITKNQPEWFRNILSIAPIMFSNKMARLGKGYMFE-CONH₂ P11: (SEQ ID NO: 15)NH2-K(X or Z)QSRMQFSSLTVNVRGSGMRILVRGNSPVFNYNK- CONH₂ P12:(SEQ ID NO: 16) NH2-K(X or Z)PDLYDYKENRFIEIGVTRREVHIYYLEKANKIKSE- CONH₂

The physiochemical properties of the native peptides are set out inTable 1 below. Residues considered to be hydrophobic are W, Y, I, F, L,V, M, A, P, G, and C. Charged residues are (+): K, R, H. (−): D, E

TABLE 1 Physicochemical Properties of Selected Peptides Positive chargesPercentage (including lysine residue Negative Peptide Hydrophobicresidues added at N-terminus) charges P1 51 10 2 P2 60 8 2 P3 60 7 2 P449 7 5 P5 60 5 4 P6 61 4 0 P7 55 6 2 P8 60 6 3 P9 60 6 2 P10 60 6 2 P1148 6 0 P12 46 9 7

Example 2: Solubility of Native Peptides and FCPs in Water

The solubility of each FCP in water was assessed. Each FCP was dispersedin 300 μl of water to a final concentration of 1.333 mM and vortexed andsonicated. The typical dispersion conditions were four sequences ofthree minutes bath sonication interspersed by 30 seconds vortexing.After inspection, the resulting solution was diluted with a mannitolsolution (a candidate lyophilisation medium, final concentration ofpeptide 0.167 mM, 1.33% (w/v) mannitol). Solubility was assessed byvisual observation of the cloudiness of the resulting dispersion(scaled: Clear/Cloudy−/Cloudy/Cloudy+) and presence of particulates. Theresults are shown in Table 2.

TABLE 2 FCP Dispersibility and Solubility in Water and 1.33% (w/v)Mannitol Solution Further dilution in Fluorocarbon- Dispersion in WaterMannitol:Water linked Peptide Solubility Solubility FCP1Cloudy/Particulates Clear/Particulates FCP2 Cloudy−/No ParticulatesClear/No Particulates FCP3 Cloudy−/Particulates Clear/Particulates FCP4Clear/No Particulates Clear/No Particulates FCP5 Cloudy/ParticulatesCloudy−/Particulates FCP6 Cloudy−/Particulates Clear/Particulates FCP7Cloudy+/No Particulates Cloudy+/No Particulates FCP8 Cloudy/ParticulatesCloudy/Particulates FCP9 Cloudy−/Particulates Clear/Particulates FCP10Cloudy−/Particulates Clear/Particulates FCP11 Clear/No ParticulatesClear/No Particulates FCP12 Cloudy/Particulates Cloudy−/Particulates

Visual inspection of the individual fluorocarbon-linked peptide solutionafter the initial dispersion in water showed that only P4 and P11 werefully soluble in water; each of these solutions was clear with nopresence of particulates. All the remaining solutions were cloudy andcontained particulates, indicating that these FCPs were not fullysoluble. On subsequent dilution with the mannitol solution, P2, P4 andP11 were fully soluble with clear solutions and no visible particulates.Solutions P1, P3, P6, P9 and P10 were also clear but particulates wereobserved, indicating that they were partially soluble in themannitol/water solution. P5, P7, P8 and P12 were insoluble in themannitol/water solution giving cloudy solutions containing particulates.Neither the percentage hydrophobicity of the peptide sequence or thepositive or negative charges was found to correlate with solubility.

For comparison, native peptide solubility was also assessed in water atthe same molecular concentration; for all native peptides except P8 thesolutions were clear with no particulates visually detected.

In conclusion, the solubility of each Fluorocarbon-linked peptide isdependent upon its aggregation properties; the majority of the FCPs werenot fully soluble in water or the mannitol solution. The solubility ofeach FCP could not be predicted from its physicochemicalcharacteristics. The equivalent native peptides were more soluble inwater than the FCPs at the same concentration.

The solubility of mixtures of the fluorocarbon-linked peptides was alsoassessed. Octavalent formulations (final concentration of each peptide0.167 mM, 1.33% (w/v) mannitol, FCP compositions provided in Table 3)were prepared. The recovery of each peptide following sterile filtration(0.22 μm Millex 25 mm PVDF filter) was determined by RP-HPLC.

TABLE 3 Composition of Octavalent Mixtures of FCPs and Recoveries ofEach FCP Following Sterile Filtration Fluoro- Fluoro- carbon- carbon-linked MIX 1 After YIELD linked MIX 2 After YIELD Peptide Blending %Peptide Blending % FCP1 Cloudy with 89.9 FCP10 Cloudy with 90.7 FCP4particulates 25.1 FCP2 particulates 97.3 FCP5 93.8 FCP4 82.3 FCP6 74.1FCP5 35.6 FCP7 31.7 FCP6 8.8 FCP3 28.6 FCP7 63.2 FCP9 22.9 FCP8 25.3FCP12 29.6 FCP11 49.4

The visual observations were confirmed by the HPLC filtration recoveryresults. The total RP-HPLC filtration recovery yields were approximately50% and 57% for MIX 1 and MIX 2 respectively indicating that largeparticulates of FCPs were removed upon filtration. Mixtures of FCPs aretherefore also poorly soluble in water.

Example 3: Solubility of FCPs in Excipients and Dispersants

In order to improve the solubility of fluorocarbon-linked peptides inwater a range of excipients and dispersants, that have proved beneficialpreviously in pharmaceutical product manufacture, were evaluated. Theseincluded Polyethylene glycols, Pluronic surfactants, lecithin, glycerin,soybean oil, safflower oil, glycofurol, dipalmitoyl phosphatidylcholine,Labrafac CC (a medium-chain glyceride), hydroxyl propyl betacyclodextrin(HPBCD) and sulfobutyl ether betacyclodextrin and combinations thereof.The solubility of a heptavalent equimassic mixture offluorocarbon-linked peptides was determined by microscopic inspection(final concentration 2.5 mg/ml).

None of the conditions tested was able to achieve a good dispersion ofthe fluorocarbon-linked peptides. HPBCD was found to improve thesolubilisation but one day incubation at room temperature was needed toachieve 70-80% solubility. In conclusion, the fluorocarbon-linkedpeptides were resistant to the action of dispersants such ascyclodextrins, surfactants or block-copolymers.

Example 4: Solubility of FCPs in Organic Solvents

The solubility of fluorocarbon-linked peptides was assessed in a rangeof organic solvents. For 80% (v/v) propan-2-ol, tert-butanol, DMSO andacetone, solutions were prepared to the same fluorocarbon-linked peptidefinal concentration of 1.33 mM. For 80% (v/v) acetic acid the finalconcentration of the fluorocarbon-linked peptide was 2.0 mM. The resultsare presented in FIG. 3.

In conclusion, all fluorocarbon-linked peptides were soluble in 80% v/vacetic acid, with no foam or particulates observed. The 80% (v/v)propan-2-ol, tert-butanol, DMSO and acetone solutions were not able toprovide complete solubility for the FCPs evaluated.

Example 5: Effects of Mannitol on Solubilisation

The effect of dilution and addition of mannitol on solubilisation invarious solvents was investigated. Each fluorocarbon-linked peptide wasdispersed in 80% v/v solvent in water according to the FIG. 4 andvortexed, followed by a seven-fold dilution with a mannitol solution.For 80% v/v propan-2-ol, tert-butanol, DMSO and acetone solutions wereprepared to the same fluorocarbon-linked peptide final concentration of0.167 mM and a final mannitol concentration of 1.33% (w/v). For 80% v/vacetic acid the final concentration of the fluorocarbon-linked peptidewas 0.25 mM. The results are presented in FIG. 4.

Equimolar mixtures of fluorocarbon-linked peptides were also prepared asabove containing the following peptides in each solvent:

Mix 1: FCP1, FCP3, FCP4, FCP5, FCP6, FCP7, FCP8, FCP12.

Mix 2: FCP2, FCP4, FCP5, FCP6, FCP7, FCP8, FCP10, FCP11.

Effective solubility of the individual FCPs and Mix 1 and 2 was onlyachievable using 80% (v/v) acetic acid as presented in FIG. 4.

Example 6: Recovery of FCPs Following Sterile Filtration of FCPSolutions

The recovery following sterile filtration (0.22 μm Millex 25 mm PVDFfilter) of each fluorocarbon-linked peptide in the mixtures prepared inExample 4 was determined by RP-HPLC. The results are shown in Tables 4and 5 below.

TABLE 4 % Filtration Recoveries of Individual FCPs From OctavalentMixture Mix 1 80% v/v Fluorocarbon- 80% v/v 80% v/v Tert- 80% v/v 80%v/v linked peptide Acetic acid Propan-2-ol butanol DMSO Acetone FCP1100.2 97.8 96.1 95.1 89.9 FCP12 100.2 16.9 6.6 32.0 12.7 FCP3 100.5 97.9102.0 100.8 99.6 FCP4 99.8 20.5 19.3 77.2 24.4 FCP7 99.4 90.9 99.9 47.842.7 FCP9 99.8 84.3 84.9 87.7 69.1 FCP5 100.2 34.3 90.2 17.2 3.2 FCP6101.3 78.4 62.3 85.8 63.2 Mean 100.2 65.1 70.2 67.9 50.6

TABLE 5 Percentage Filtration Recoveries of Individual FCPs From theOctavalent Mixture Mix 2 80% v/v Fluorocarbon- 80% v/v 80% v/v Tert- 80%v/v 80% v/v linked peptide Acetic acid Propan-2-ol butanol DMSO AcetoneFCP11 99.5 57.4 52.3 85.4 76.4 FCP2 99.8 92.7 99.5 98.2 85.4 FCP4 10021.6 10.0 73.0 4.5 FCP7 93.3 90.5 89.2 46.4 43.7 FCP8 99.8 20.7 21.234.9 17.6 FCP10 98.1 87.7 87.3 89.4 79.4 FCP5 99.1 35.9 51.8 18.1 2.9FCP6 97.4 69.5 82.1 91.0 64.1 mean 98.4 59.5 61.7 67.1 46.7

No loss of fluorocarbon-linked peptide was detected following sterilefiltration for the mixtures prepared using 80% (v/v) acetic acid. It isconcluded that acetic acid with subsequent aqueous dilution is thepreferred solvent for the dissolution and filtration offluorocarbon-linked peptides, but it will be necessary to reduce theconcentration used in order to minimise the levels of residual aceticacid in the final product.

Example 7: Effect of Concentration of Acetic Acid on Solubility of FCPs

In order to limit the concentration of acetic acid downstream in theformulation process and in the final product, the lowest concentrationof acetic acid in water to achieve efficient dispersion and maintainvisible solubility was determined for each fluorocarbon-linked peptide.When using mannitol as a cryprotectant, it is important to minimize theacetic acid concentration at the lyophilisation stage in order toachieve a stable and amorphous freeze-dried product. The minimum aceticacid concentration to achieve acceptable dispersibility and solubilityof the individual fluorocarbon-linked peptides was determined. The finalconcentration of fluorocarbon-linked peptide after initial acetic aciddispersion was 2 μmol/ml and after dilution with mannitol, 0.250μmol/ml.

TABLE 6 Dispersibility and Solubility of Individual FCPs and aHeptavalent Mixture (Mix 3) in Acetic Acid Visual Percentage Ease ofVisual appearance of Fluorocarbon- acetic dispersion by appearancemixture after linked acid sonication/ after dispersion: peptide (% v/v)vortexing dispersion Mix 3 FCP1 10 +++ Clear Clear FCP2 10 +++ ClearFCP4 10 +++ Clear FCP8 80 + Clear FCP9 80 + Clear FCP5 10 +++ Clear FCP680 + Clear

A concentration of acetic acid as low as 10% v/v was found to provideadequate dispersion for several of the fluorocarbon-linked peptides.However, whilst some fluorocarbon-linked peptides required less than 80%(v/v) acetic to achieve full dissolution, the resulting formulationproved to be physically unstable over time with a gel or solubleparticulates being formed (for example FCP6, FCP8 and FCP9). For thesethree peptides, 80% (v/v) acetic acid was found to achieve completedispersion while preventing any change in physical state.

Filtration recovery was measured by RP-HPLC comparing peak areas of eachfluorocarbon-linked peptide within the Mix 3 mixture before and aftersterile filtration. The percentage recovery was measured for each FCPand a mean recovery calculated as an average of the percentage ofrecovery of each individual FCP. The overall filtration recoveries(based on 0.22 μm filter) measured after blending and dilution weretypically greater than 95%.

TABLE 7 Filtration Recovery FCP Filtration recovery FCP1 100.0 FCP2 99.3FCP4 98.6 FCP5 98.7 FCP6 100.0 FCP8 97.0 FCP9 95.4 Mean 98.4

Example 8: Characterisation of Structures Formed by Fluorocarbon-LinkedPeptides

The formation of self-assembled multimolecular micellar structures mayplay a central role in the solubilisation process of fluorocarbon-linkedpeptides. In this manner, the solubility of fluorocarbon-linked peptidescan be maintained following dispersion and subsequently throughout theformulation process, particularly in the reconstitution of thelyophilised product. The physical characterisation of the multimolecularstructures assembled during dispersion was performed using Dynamic LightScattering (DLS) and Transmission Electron Microscopy (TEM).

A Zetasizer Nano S (enabling measurement of particles from 0.6 nm to 6microns) was employed to monitor the particle size of a mixture offluorocarbon-linked peptides based on DLS. Mix 1 (FCP1, FCP3, FCP4,FCP5, FCP6, FCP7, FCP9 and FCP12) was prepared as described in Example 1(Table 3) with mannitol diluent.

The average particle size (nm) of each mixture was measured at 25° C.using a Nanosizer (Zetasizer Nano Series ZS, Malvern Instruments, UK).250 μl of solution was used and dispatched in a plastic microcuvette.Correlation times were based on 10 s per run and a total of 5 runs permeasurement were made. Results were analysed using Dispersion TechnologySoftware (Malvern Instruments, UK). Size distribution by volume and inintensity was obtained for Mix 1. Average size based volumetricmeasurement was calculated by the Dispersion Technology Software.

The DLS of fluorocarbon-linked peptides in solution demonstrates thepresence of multimolecular structures of diameter centred around 20 to50 nm (>95% by volume) with approximately 12-16% (by intensity) of thepopulation with a size greater than 100 nm (FIG. 5).

TEM showed the presence of a homogenous population of sphericalstructures of dimensions consistent with the DLS (FIG. 6).

Example 9: Impact of Sterilising Grade Filtration on Multimolecular FCPStructures

The impact of sterilising grade filtration upon the multimolecularstructures was investigated. Mix 1 from Example 7 was filtered via asterile 0.22 μm Millex 25 mm PVDF filter and then analysed by DLS usinga Zetasizer Nano S as described in Example 7.

DLS analysis demonstrated that the structures formed are highly dynamicwith variable reproducibility even within the one set of analysis.Between five and seven measurements were collected to calculate theaverage particle size (represented by the different profiles in FIG. 7).

Surprisingly, it was found that the 0.22 μm filtration may re-shape themultimolecular structures formed by fluorocarbon-linked peptidesinitially solubilised in acetic acid. DLS shows that large particles arere-shaped into smaller particles post-filtration and that the Kcount (aparameter that correlates with the number of particles in solution) isalso drastically reduced post-filtration (pre-filtration, 200 Kcounts;after post-filtration, 120Kcounts). Moreover, the introduction of a 0.45μm filter ahead of the 0.22 μm filter did not reduce filtrationrecoveries. Sterilising grade filtration can therefore influence theresulting size of the structures assembled, not by simply removing largeparticles from the formulation and restricting their passage downstreamof the manufacturing process (with a concomitant reduction in yield),but rather by re-shaping the structures by deformation so they are ableto pass through the filter. Particles with size over 220 nm representaround 12 to 16% (by intensity) of the particles in the mixture,according to the DLS data. These would appear not to be removed from thesolution as the filtration recovery determined by HPLC is over 97%.

The MIX 1 formulation particle size distribution was also assessed afterfreeze-drying following reconstitution of samples in Tris-HCl 10 mM orwater (FIG. 8). The MIX 1 formulation was readily reconstitutedachieving a clear or slightly opalescent solution with water or Tris-HCl10 mM respectively. Particle sizes were centered around 20-50 nM with aprofile broadly similar to that observed during formulation(pre-lyophilisation). This demonstrates that post-reconstitution; FCPsmultimolecular structures are maintained without the formation of largevisible aggregates.

Example 10: Chemical Stability of FCPs

The chemical stability of the fluorocarbon-linked peptides was assessedby exposing a lyophilised formulation of seven fluorocarbon-linkedpeptides to 50% (v/v) acetic acid for 24 hours. Mix 3 was prepared byinitial solubilisation of the FCPs in acetic acid (concentration ofsolvent for each FCP as given in Example 6), followed by dilution andblending with a mannitol solution. The mixture was then lyophilisedprior to reconstitution in 50% (v/v) acetic acid.

No degradation was observed by RP-HPLC compared to an untreated control(see FIG. 9). This demonstrated that the selected fluorocarbon-linkedpeptides are chemically stable in 50% v/v acetic acid for the durationof a typical blending step during a pharmaceutical manufacturingprocess.

Example 11: Residual Acetate Concentration in Final Presentation

It is important to minimise the acetic acid concentration in thedownstream formulation. This will allow the formation of a stable cakeduring lyophilisation and raise the pH of the preparation closer to thedesired neutrality. Acetic acid is volatile and its content therebyreduced during the freeze-drying process.

For lyophilisation, formulations of Mix 3 prepared as described inExample 9 (3 ml freeze-drying vials filled with 1.4 ml volume) werefirstly frozen for two hours in an −80° C. freezer and then freeze-dried(benchtop Christ Alpha2-4 LSC) for 24 hours. This procedure allowed theproduction of lyophilised cakes with a stable structure and homogenousconsistency. The pre-lyophilisation concentration of acetate in theformulation was calculated to be 8.8% v/v.

For three different batches, the post-lyophilisation residual acetateconcentration (acetate counterions plus acetic acid) in the vials wasexperimentally determined to be 0.3-0.4, 0.7 and 0.5% (w/w)respectively. The standard deviation of this analysis was validated as+/−0.07% (w/w) for acetate; the limit of quantitation as 0.1% (w/w). Themean value of residual acetate equates to an acceptable level ofapproximately 0.35 mg per human dose, well below the ICH recommendation(maximum 50 mg per day).

Example 12: Reconstitution of Lyophilised FCP Preparations

The reconstitution of formulated fluorocarbon-linked peptides(Heptavalent, Mix 3) was compared to an unformulated equivalentpreparation. The individual FCPs were solubilised in acetic acid asdescribed in Example 9, blended and diluted with a mannitol solution andlyophilised (final concentration 0.35 mg per FCP). One vial of theformulated mixture was reconstituted with 0.7 ml of Water for Injection(Hyclone); an additional vial was reconstituted with 0.7 ml of 28 mMhistidine buffer solution. For the unformulated mixture 0.35 mg of each,untreated, FCP was dispensed into a vial with no additional processing.One vial of the unformulated mixture was reconstituted with 0.7 ml of4.5% mannitol solution to provide an identical excipient concentrationto that of the formulated vials. An additional vial of the unformulatedmixture was reconstituted with 0.7 ml of 28 mM histidine in 4.5%mannitol solution.

The photographs below (FIGS. 10 and 11) illustrate the solubility of theFCPs following initial dispersion by manual shaking and subsequentvortexing for 30 seconds and bath sonication for a period of one hour.

Visual inspection of the unformulated samples after reconstitution withboth the mannitol/water and histidine buffer solutions showed that thesolutions were not fully dispersed and solubilised. Each solution wascloudy and large particulates could be observed, in particular adheringto the side of the glass vial, which did not disperse over time. Incontrast, the solutions of the formulated vaccine dispersed in both thewater and histidine buffer solutions were clear with no presence ofparticulates.

These results demonstrate that the method of initial solubilisation hasan impact upon the aggregative properties of the final reconstitutedformulation. Dispersion of the FCP in acetic acid early in theformulation process directs the formation of micellar structures, whichare maintained through the subsequent blending, filtration andlyophilisation processes. This facilitates the reconstitution of thefinal lyophilised presentation in an aqueous phase in a non-particulateform.

Conclusions

Acetic acid was demonstrated to be a good solvent for all individualfluorocarbon-linked peptides evaluated. Complete solubilisation wasachievable at high FCP concentrations (up to 2000 nmol/ml; approximately10 mg/ml) with no particulates observed following perturbation byvortexing and sonication. Solubility was maintained during furtherdownstream processing due to the amphiphilic properties offluorocarbon-linked peptides directing the formation of spontaneouslyself-assembled macromolecular structures as observed by Dynamic lightscattering and transmission electron microscopy. The solvent istherefore contributing a dual role; firstly in ensuring that there issufficient disruptive capability to ensure that large disorderedparticulate structures are disrupted and secondly supporting anenvironment whereby multimolecular, ordered, micellar structures may becreated and supported. These structures are small enough to allowsolubilisation of the FCP such that no loss of material occurs uponsterile filtration. The micellar structures are also retained duringlyophilisation and are essential to facilitate the resolubilisation ofthe lyophilisate in an aqueous media prior to administration to humans.FCPs that had not been previously solubilised in acetic acid could notbe satisfactorily reconstituted from a freeze-dried state in water orhistidine buffer (Example 12).

80% (v/v) acetic acid was found to solubilise all FCPs. However,excessive acetic acid can prevent the formation of an acceptablelyophilisate cake following freeze-drying. In addition, there areregulatory constraints imposed upon the levels of acetate inpharmaceutical products. Lower concentrations of acetic acid weretherefore examined; with 10% (v/v) proving to be suitable for four ofthe seven FCPs, whilst 80% (v/v) was the lowest concentration viable forthe remaining three FCPs. On processing, this blend of seven FCPsproduced an acceptable lyophilisate cake with compliant levels ofacetate per human dose.

All other solvents investigated were unable to achieve completesolubilisation of all the FCPs. The success of acetic acid was notpredictable, as the fluorocarbon chain imparts unusual physicochemicalproperties upon the molecule (compare for example, the solubilities ofthe FCPs and the equivalent native peptides in Example 1). In addition,there was no correlation between the success of 10% (v/v) acetic acid insolubilising an FCP and the hydrophobicity or charged residue content ofthe peptide.

In summary, acetic acid has the following advantages:

capable of providing adequate solubility for not only the individualFCPs but also mixtures thereof;

suitable for all FCPs evaluated;

Yields consistent and uniform products;

Water-miscible at the concentrations intended for use (10-80% (v/v);

Able to solubilise the FCPs at relatively high concentrations (at least10 millimolar);

Listed as a ICH class III solvent, suitable for human use;

Amenable to lyophilisation (with levels being reduced after a typicalfreeze-drying stage);

Results, after subsequent blending and dilution, in a solution that vanbe subjected to sterilising grade filtration with minimal yield losses;

Results, after lyophilisation, in a product that can be readilyreconstituted to form an isotonic, pH neutral, homogeneous suspension;

Does not react with, or promote degradation of, the fluorocarbon-linkedpeptide; and Compatible with the materials routinely used inpharmaceutical product manufacture.

Example 13: Preparation of a Fluorocarbon-Linked Peptide InfluenzaVaccine

The objective of this study is to demonstrate the benefit of aformulation process designed for the good manufacturing practice (GMP)production of a pharmaceutically acceptable universal influenza-Avaccine (FP01.1) containing six fluoropeptides, which comprises peptideswith the amino acid sequences shown in SEQ ID NOs: 1 to 6. The specificobjectives are:

1. To assess key formulation parameters for the manufacture of FP-01.1.

a. Ease of fluoropeptide solubilisation in acetic acid solutions;

b. Micelle size determination at the point of filtration;

c. Filtration recovery;

d. Chemical and physical stability of the fluoropeptides; and

2. To compare the quality of the reconstituted FP-01.1 vaccine(formulated fluoropeptides) with an equivalent preparation containingnon-formulated fluoropeptides.

We have developed a formulation process and applied it to themanufacture of a universal influenza-A vaccine FP-01.1 composed of sixfluorocarbon-linked peptides comprising SEQ ID NOs: 1 to 6,respectively. The six peptides having the sequences shown in SEQ ID NOs:1 to 6 are coupled to the fluorocarbon chain C₈F₁₇(CH₂)₂COOH via theepsilon chain of an N-terminal lysine spacer. The sixfluorocarbon-linked peptides thus correspond to FCP1, FCP8, FCP9, FCP2,FCP4 and FCP5 described in the preceding Examples. The formulationprocess is based on the use of acetic acid, an acidic solvent that wehave found ensures good dispersability of the fluoropeptides whilstmaintaining physical and chemical stability of the fluoropeptides duringthe process. Acetic acid is highly volatile and can be sublimated duringfreeze-drying and we have found that it is consistently reduced toresidual levels that have little impact on the pH of the reconstitutedproduct.

The formulation process described below achieves the manufacture of afreeze-dried FP01.1 vaccine (ensuring long term stability) to bereconstituted with a buffer solution (28 mM L-Histidine) to generate astable homogenous solution (no visible aggregates) with neutral pH(6-7.5) and acceptable osmolality (280-320 mOsm). Several GMP clinicalbatches have been usefully produced and we have demonstrated that theproduct is safe and immunogenic in humans.

Materials and Instruments

Fluoropeptides (contained in FP-01.1) manufactured by the AmericanPeptide Company

Glacial acetic acid (Sigma#27225), D-Mannitol (Merck Emprove)

Hyclone water (Fisher#HYC-001-189G)

Millex, PVDF Durapore, 0.2 μm (033 mm) Millipore.

30 Autoclaved (Freeze dried vials (Adelphi#VC002-13C)+stoppers(Adelphi#FDW13)).

HPLC equipped with Discovery Column C18, 250×2.1 mm, 5 μm

Combitips pipette tips 10 ml (Fisher#PMP-117-523N)+Eppendorf Stepper

Freeze drier 2-4-LSC (Christ)

Osmomater: Osmomat 030 (Gonotec)

pH meter equipped with micro Inlab® electrode (Mettler)

Methods Solution Preparation

1. 3.3% w/w Mannitol in 50 ml in water (6.6 g in 200 ml water), cooldown at 4° C.2. 5 ml solutions of acetic acid at 10% (v/v) in sterile water.3. 5 ml solutions of acetic acid at 80% (v/v) in sterile water.

Fluorocarbon-Linked Peptide Weighing

TABLE 8 FCPs weighing and dispersion Peptide Mass (mg) Acetic Vol (μl)of Vol (μl) of Mass (mg) content Theoret. Mass (mg) Acid Conc aceticacid- acetic acid- FCP (net) (%) (gross) Experim. (%) Theoret. Exper.FCP1 10 87.0 11.49 12.27 10 1000 1068 FCP2 10 87.6 11.41 12.49 10 10001094 FCP4 10 90.6 11.04 11.68 10 1000 1058 FCP5 10 92.0 10.87 11.67 101000 1074 FCP9 10 90.3 11.07 11.60 80 1000 1048 FCP8 10 92.0 10.87 11.7880 1000 1084

Formulation Preparation for FP-01.1

-   -   1. Weigh each peptide (targeting 10 mg net peptide) in a 2 ml        glass vial    -   2. Disperse each fluoropeptides at 10 mg net/ml in ˜1.0 ml        (adjust volumes in function of weighing to get exactly 10 mg        net/ml) of 10% or 80% acetic acid solution in water (see table        10),    -   3. Vortex and sonicate and record visual aspect    -   4. Repeat step 3 until complete dissolution    -   5. Blend together 950 μl of each of the 6 dispersed        fluoropeptides into a 40 ml glass container. Then add 950 μl of        acetic acid 80% (6.65 ml total volume). Each peptide is at a        concentration of 1.428 mg/ml in 40% acetic acid.    -   6. Record visual aspect of the blended solution    -   7. Dilute the blended fluoropeptides with 25.93 ml of 3.3%        mannitol (solution at 0.2915 mg/ml of each peptide), total        acetic acid 8.16%.    -   8. Record visual aspect of the diluted solution    -   9. Filter the ˜32 ml solution with a Millex PVDF 33 mm, 0.2 μm        (keeping 0.3 ml unfiltered solution for filtration recovery).

Filling

Aliquot labelled 2 ml freeze according to Table 9 (Filling volume: 1.2ml for each formulation), using 10 ml combitips.

TABLE 9 Preparation of Fluoropeptides formulation FP-01.1 PeptideAliquot Peptide Buffer Vol. Final Total conc. for Lyo- quantity forrecon- concentration Volume (mg/ philisat (μg/vial/ stitution afterrecon- (ml) pept/ml) (μl) peptide) (μl) stitution 29-30 0.2915/ 1200 350700 0.500 ind (24-25 mg/pept/ml vials)

Freeze Drying

-   -   1. Freeze the vials at −80° C. for one hour.    -   2. Freeze dry for 40 hours    -   3. Freeze drying ventilation is performed under nitrogen and the        vials stoppering is carried out at a pressure between 400 and        600 mbar.

Preparation of FP-01.1-Equivalent Using Non Formulated Fluoropeptides

Two vials were prepared containing 0.35 mg of each of the 6fluoropeptides: FCP1, FCP8, FCP9, FCP2, FCP4 and FCP5.

Transmission Electron Microscopy

TEM in negative staining, 20 μl of fluorocarbon-linked peptide solutionis deposited on a Formvar carbon coated copper electron microscope grid(300 mesh). 20 μl of uranyle acetate (1% aqueous) is then added. After30 seconds, excess solution is quickly wicked away with a Whatman filterpaper. The sample is then allowed to dry for at least 2 minutes beforeanalysis. Transmission electron microscopy is then performed on PhilipsCM120 biotwin at 120 kV accelerating voltage. Image acquisition isperformed at a direct magnification ranging from 50000× to 150000×.

RP-HPLC Analysis

HPLC Method: FP-01.1

-   -   Column: Discovery C18: 2.1×25 mm, 5 μm, Flow 0.3 ml/min.    -   Solvent A: 90% water/10% acetonitrile/0.04% TFA    -   Solvent B: 90% acetonitrile 10% water 0.04% TFA).    -   Gradient:

Time (min) Solvent B 0.01 10% 1 10% 6 28% 46 44% 50 57% 60 70% 68 82% 7082% 71 10%

Results Formulation Step (Before Freeze Drying) Peptide Dispersion

TABLE 10 Ease of solubility of each peptide after vortex/sonicationcycle Cycle 1 Cycle 2 Cycle 3 FCP Vortexing Sonication VortexingSonication Vortexing Sonication FCP1 Clear Clear Clear Clear Clear ClearFCP2 Clear Clear Clear Clear Clear Clear FCP4 Clear Clear Clear ClearClear Clear FCP5 Clear Clear Clear Clear Clear Clear FCP9 Particulates−Clear Clear Clear Clear Clear FCP8 Particulates+ Some Some 1 or 2 1 or 2Clear particulates particulates particulates particulates

Peptides were easily soluble with no sonication required, whereas thetwo peptides soluble in acetic acid 80% required at least 1 cycle ofsonication.

Blending/Dilution

The solution of the 6 peptides blended was clear (no visibleaggregates). Once diluted with the mannitol at 3.3%, the solution wasstill clear (no visible aggregates).

TABLE 11 Visual Appearances of Preparations Visual Visual PercentageEase of Visual appearance appearance acetic dispersion appearance ofmixture of mixture Pep- acid (see Table after after after tide (% v/v)12) dispersion blending Dilution FCP1 AcOH +++ Clear Clear Clear 10%FCP2 AcOH +++ Clear 10% FCP4 AcOH +++ Clear 10% FCP5 AcOH +++ Clear 10%FCP9 AcOH ++ Clear 80% FCP8 AcOH + Clear 80%

Filtration Recoveries

Very good filtration recoveries were achieved (>99% for each peptide).

TABLE 12 Peptide recoveries after filtration FCP FCP1 FCP2 FCP4 FCP5FCP9 FCP8 Peptide recovery % 100.2 101.6 100.8 100.2 99.8 99.6

Transmission Electron Microscopy (TEM) Imaging

TEM analysis of the micelles formed before filtration shows the presenceof a homogenous population of small spherical micelles with a sizeranging from 17-30 nm (FIG. 12).

Chemical Stability (Post-Filtration)

Chemical stability was determined by RP-HPLC at T₀ and after 24 hourspost-filtration. The results are shown in FIG. 13 and Table 13.

TABLE 13 Post-filtration purities over time T0 2 hrs 8 hrs 24 hrs Purity% 96.9 97.1 97.1 96.9 HPLC file 8644 8645 8649 8651

Analysis Performed on Finished FP-01.1 Product (Post-Freeze-Drying)

Cake Aspect after Freeze Drying

TABLE 14 Cake inspection results Elegant Collapsed Total Product cakecake vials FP01.1 25 0 25The freeze-dried product forms an elegant solid uniform cake.

Purity Analysis of Freeze-Dried FP-01.1 Product

A sample was reconstituted in 0.70 ml water to get a concentration at0.5 mg/peptide. No degradation occurred during the freeze drying, withthe purity of 97%.

Reconstitution of Freeze-Dried FP-01.1—Comparison with UnformulatedFluoropeptides

To demonstrate the benefit of the formulation process applied to theproduction of FP-01.1, the quality of the reconstituted FP-01.1 vaccine(formulated fluoropeptides) was compared with an equivalent preparationcontaining non-formulated fluoropeptides.

The FP-01.1-equivalent based on non-formulated fluoropeptides wasprepared by weighing the 6 fluoropeptides in a single vial (0.35 mg ofeach peptide). The non-formulated FP-01.1-equivalent was reconstitutedwith either 0.7 ml of water (containing 4.5% mannitol to be equivalentto FP-01.1) or 28 mM L-Histidine (containing 4.5% mannitol) and comparedwith the formulated FP-01.1 (obtained through the formulation processdescribed above) and reconstituted under the same conditions. Thereconstituted formulated FP01.1 was analysed by RP-HPLC and the resultsof the analysis are shown in FIG. 14.

The formulated FP-01.1 product was easily reconstituted in water whilethe non-formulated FP-01.1 equivalent is insoluble with large aggregatesin suspension and adhering to the glass wall (see FIG. 15). FP-01.1reconstituted in water lead to a very slightly opalescent homogeneoussolution with no visible aggregates. The non-formulated fluoropeptidesdo not achieve solubility over time and even after sonication andvortexing.

Similarly, the formulated FP-01.1 product was easily reconstituted in 28mM L-Histidine (buffer designed for the clinical product to achieveneutral pH) while the non-formulated peptides are insoluble (formationof large aggregates) (see FIG. 16). FP-01.1 reconstituted in water leadto a slightly opalescent homogeneous solution with no visibleaggregates. The non-formulated fluoropeptides do not achieve solubilityover time even after sonication and vortexing.

Based on these results, a product based on the non-formulatedfluoropeptides would not be considered to be pharmaceutically acceptabledue to its poor dispersability and the absence of homogeneity of thepreparation.

Osmolality and pH of the Formulation in L-Histidine

TABLE 15 pH and osmolality of the reconstituted formulation inL-Histidine FP-01.1 reconstituted in Histidine 28 mM Osmolality 302(mOsm/kg) pH 6.65

Conclusions

-   -   All fluoropeptides achieved full solubility at the point of        dispersion.    -   Micelles were formed with a size ranging from 17 to 30 nm        compatible with sterile filtration (220 nm cut-off).    -   Sterile Filtration recovery was over 99% for all fluoropeptides.    -   FP-01.1 was easily reconstituted with its dedicated 28 mM        L-histidine buffer systems leading to a homogenous slightly        opalescent solution with close to neutral pH and acceptable        osmolality (˜300 mOsm).    -   An FP-01.1-equivalent preparation obtained from non-formulated        fluoropeptides was demonstrated to be difficult to reconstitute        with fluoropeptides forming large insoluble aggregates with a        large proportion adhering to the glass wall. This contrast with        the reconstitution of a formulated FP-01.1 and demonstrate the        benefit of the formulation process in generating a        pharmaceutically acceptable product.

Example 14: Immunogenicity of FP01.1 in Rats

The immune response that can be generated by the FP-01.1 formulation wasassessed in rats were immunised intramuscularly on the lower left flankby staff members of the CBS, St Mary's Campus, Imperial Collegeaccording to GD_RD004.

Preparation of Splenocytes

Rats were sacrificed according to home office regulations and GD_RD004.Spleens were harvested (any abnormal spleens were photographed) andsingle cell suspensions were prepared according to GD_RD007, with cellnumbers determined as described in GD_RD001 (TruCount Method).Splenocytes were resuspended to 1×10⁷/mL in complete media and platedfor IFNγ ELISpot and CBA.

IFNγELISpot

Antigens for stimulation were freshly prepared at 4× concentration fromstocks and plated in duplicate wells for IFNγ ELISPOT. Cells were platedat 0.5×10⁶ splenocytes/well (504, of cell suspension), with 504, of 4×antigen preparation (i.e. individual long peptides and LPMIX6 forstimulation) and 1004, of complete media (total volume=200 μL). ELISpotplates were incubated for 18 hours at 37° C., 5% CO₂ in a humidifiedenvironment.

Three doses of the FP-01.1 were adjusted by volume maintaining aconstant FCP concentration of 500 μg/ml per peptide. SD rats wereinjected IM with 12.5, 50 or 100 μg/peptide of FP-01.1 on days 0 and 14and their spleens harvested on day 24 for IFN-γ ELISPOT analysis after18 hours incubation with individual native peptides from FP-01.1. Both12.5 μg/peptide and 50 μg/peptide doses were injected at a single sitein a volume of 25 μl and 100 μl respectively, while the 100 μg/peptidedose was injected in 2×100 μl volumes at two different sites.

FP-01.1 induced a positive IFN-γ T cell response at all dose levelstested in a dose dependent fashion (FIG. 17).

Example 15: Clinical Trial Data for FP01.1

Three ascending doses of FP-01.1 (50, 150, 500 μg/peptide) and placebogiven on days 1, 29 was assessed for safety, tolerability andimmunogenicity in a phase I clinical trial in a total 48 healthyindividuals.

FP-01.1 was well tolerated by all three cohorts, following twointramuscular injections. There was no clear evidence of adose-dependent relationship in the incidence of TEAEs, laboratoryparameters or injection site reactions. No subject exhibited any markedlocal or systemic reaction to vaccination on either the first or secondexposure in any of the three cohorts.

Vaccine-induced T cell responses were assessed using an ex vivo IFN-γELISpot assay. PBMCs were stimulated with 6 individual peptides(corresponding to peptides contained in the vaccine) for 18 hours.Positive assay responses were defined as the mean of number of spots inthe negative control wells+2 standard deviations of the mean. The numberof spots for each of the 6 peptides was cumulated to obtain the “sum forlong peptides” and expressed as a number of spots per million inputPBMCs.

FP-01.1 was demonstrated to be immunogenic. 150 μg FP-01.1 dose groupshows a higher response compared with the two other vaccine doses andthe placebo group as observed in FIG. 18. In this dose group, a boostereffect is observed after the second injection supporting the concept ofbooster amplification with multiple injections for peptide vaccines.

1-27. (canceled)
 28. A sterile, dried mixture of fluorocarbon-linked peptides, wherein the dried mixture comprises one or more fluorocarbon-linked peptides comprising 20 to 50 amino acid residues linked to a fluorocarbon, at least 50% hydrophobic amino acid residues, an isoelectric point greater than or equal to 7, and one or more T cell epitopes, not comprising a contiguous sequence of 20 amino acid residues comprising more than 80% hydrophobic amino acid residues; and wherein the mixture is in a solid form.
 29. The dried mixture of claim 28, wherein the solid form is lyophilized as an amorphous cake or a powder.
 30. The dried mixture of claim 28, wherein the solid form is dried by vacuum drying, spray-drying, freeze drying or fluid bed drying.
 31. The dried mixture of claim 28, wherein the solid is freeze dried.
 32. The dried mixture of claim 28, wherein the mixture is disposed in a vial, an ampoule or a syringe.
 33. The dried mixture of claim 32, further comprising a pharmaceutically acceptable carrier.
 34. The dried mixture of claim 28, wherein the peptide linked to the fluorocarbon is an immunogenic peptide having a sequence derived from (i) a protein or peptide from a pathogen or tumor cell or (ii) an autologous protein.
 35. The dried mixture of claim 28, wherein the one or more T cell epitopes are from a pathogen, an autoimmune protein, or allergen or a tumor antigen.
 36. The dried mixture of claim 35, wherein the pathogen is a virus, bacterium, Mycobacterium, parasite or fungus.
 37. The dried mixture according to claim 28, wherein the fluorocarbon comprises a chain from 3 to 20 carbon atoms, wherein one or more fluorine moieties is optionally replaced with a halogen moiety of Cl, Br, or I; a methyl group; or a hydrogen.
 38. The dried mixture according to claim 28, wherein the fluorocarbon comprises the formula C₈F₁₇(CH₂)₂.
 39. The dried mixture according to claim 28, wherein the one or more fluorocarbon-linked peptides are present in micelles.
 40. A sterile, dried mixture of fluorocarbon-linked immunogenic peptides, wherein the dried mixture comprises one or more fluorocarbon-linked immunogenic peptides comprising 20 to 50 amino acid residues linked to a fluorocarbon, at least 50% hydrophobic amino acid residues, an isoelectric point greater than or equal to 7, and one or more T cell epitopes, not comprising a contiguous sequence of 20 amino acid residues comprising more than 80% hydrophobic amino acid residues; wherein the immunogenic peptides are derived from Hepatitis B virus (HBV); and, wherein the mixture is in a solid form.
 41. The dried mixture of claim 40, wherein the solid form is lyophilized as an amorphous cake or a powder.
 42. The dried mixture of claim 40, wherein the solid form is dried by vacuum drying, spray-drying, freeze drying or fluid bed drying.
 43. The dried mixture of claim 40, wherein the solid is freeze dried.
 44. The dried mixture of claim 40, wherein the mixture is disposed in a vial, an ampoule or a syringe.
 45. The dried mixture of claim 44, further comprising a pharmaceutically acceptable carrier.
 46. The dried mixture according to claim 40, wherein the fluorocarbon comprises a chain from 3 to 20 carbon atoms, wherein one or more fluorine moieties is optionally replaced with a halogen moiety of Cl, Br, or I; a methyl group; or a hydrogen.
 47. The dried mixture according to claim 40, wherein the fluorocarbon comprises the formula C₈F₁₇(CH₂)₂. 